Antimicrobial properties of lactic acid bacteria isolated from traditional yogurt and milk against Shigella strains

Background: Lactic acid bacteria (LAB) are normal flora of the mouth, intestines and the female genital tract. They are also frequently found in meat, vegetables, and dairy products. Most of probiotic bacteria belong to the LAB group. Some probiotic LAB are useful in prevention and treatment of diarrheal diseases. The aim of this study was to investigate the antimicrobial properties of LAB isolated from traditional yogurt and milk against Shigella strains. Materials and methods: Forty LAB strains were isolated from traditional yogurt and milk. The antimicrobial activity of LAB against Shigella strains (eight S. flexneri, four S. sonnei) was examined using the agar-well diffusion assay. LAB strains with antimicrobial effect against all Shigella strains were identified by 16S rRNA gene sequencing. Results: Six LAB strains inhibited the growth of all 12 Shigella strains. Lb. paracasei Y1-3, Lb. paracasei Y8-1 and Lb. fermentum Y2-2 were isolated from yogurt. Lb. paracasei M18-1, Lb. parelimentarius M4-3 and Lb. plantarum M19-1 were isolated from milk. Conclusion: This study showed that Lactobacillus strains with good inhibitory activity against S. flexneri and S. sonnei could be isolated from traditional yogurt and milk.


Background
Lactic acid bacteria (LAB) are normal, physiological flora of the mouth, intestines and female genital tract. They are also frequently found in meat, vegetables, and dairy products, such as milk and yogurt. The Lactobacillus genus is one of the most important genera in the group of LAB. Lactobacillus strains are Gram-positive, catalasenegative, non-spore-forming and non-motile bacilli [1], [2], [3]. LAB have protective effects in fermented food preservation, because they produce organic acids in food during their growth. Conversion of carbohydrates to organic acids and reduction of pH is the reason for the increased half-life and good quality of such food products [1], [4]. Probiotics are living microorganisms which, when consumed in adequate amounts, confer health benefits to the host by altering the indigenous microflora [5]. Most probiotic bacteria belong to the LAB group [6]. The presence of LAB in food has beneficial effects on human health, including effects on the natural gut microflora equilibrium, reducing blood cholesterol, decreasing intestinal tumors, facilitating calcium absorption in the intestines, reducing lactose intolerance, and preventing and treating diarrheal diseases [7], [8], [9]. Several mechanisms have been suggested for the inhibitory activity of LAB against pathogenic bacteria, especially Gram-negative pathogens. These mechanisms include production of organic acids, hydrogen peroxide and bacteriocin, and competition for colonization sites with pathogenic bacteria [10], [11], [12]. Shigella spp. are common intestinal Gram-negative pathogens which cause diarrheal diseases and dysentery in many countries [13]. Shigella spp. are a leading cause of gastroenteritis-induced deaths in 3-5 million children under five years old in developing countries [14]. In several studies, the probiotic potential of various Lactobacillus strains has been demonstrated. Some Lactobacillus strains commonly used as probiotics are efficaceous especially against acute diarrhea in children [11], [15], [16], [17]. The purpose of this study was to investigate the antimicrobial effect of LAB isolated from traditional yogurt and milk against Shigella spp.

Samples collection and culture
Twenty samples of traditional yogurt and milk were collected in sterile containers and transferred to the microbiology laboratory of Babol University of Medical Sciences, Iran. Two grams of yogurt and 500 µl of milk were inoculated into 15 ml de Man, Rogosa and Sharp (MRS) broth medium (Merck, Germany), and cultured for 48 h in anaerobic jars at 37°C. Then the MRS broth was subcultured on MRS agar (Merck, Germany) plates, and inoculated for 48 h in anaerobic jars at 37°C. The suspicious colonies were tested using Gram staining and catalase reaction. Gram-positive and catalase-negative bacteria were purified by streaking on MRS agar, and stocked in MRS broth containing 20% glycerol at -20°C.

Evaluation of antimicrobial effect of LAB against Shigella strains
The antimicrobial activity of LAB against Shigella strains was tested using the agar well diffusion assay [18]. Twelve Shigella strains (eight S. flexneri, four S. sonnei), which were previously isolated from children with diarrhea, were included in this study. The LAB were cultured in 3 ml MRS broth medium in anaerobic jars and incubated for 24 h at 37°C. The MRS broth tubes were subsequently centrifuged (10,000 g, 10 min) to prepare cell-free culture supernatants (CFCS). Shigella strains were grown on nutrient agar medium (Merck, Germany) and incubated for 24 h at 37°C. A suspension of 10 7 colony forming units (CFU)/ml of Shigella strains was then prepared and spread onto the nutrient agar, into which 5-mm-deep wells had been dug. About 100 µl of CFCS was poured into each well, and nutrient agar plates were incubated for 18 h at 37°C. Lb. rhamnosus GG was used as the positive control. Finally, inhibition zone diameter was measured. LAB strains with clear zones <11 mm, 11-16 mm, 17-22 mm and >23 mm, were classified as negative (-), mild (+), strong (++), and very strong (+++) inhibitors, respectively.

Production of bacteriocin-like compounds
Two main mechanisms of antimicrobial activity are the production of organic acids, which reduce the pH, and the production of hydrogen peroxide. Some Lactobacillus spp. also produce bacteriocin, which is antimicrobially active [19]. For these reasons, the pH of the CFCS was measured and adjusted to 6.5 with NaOH (Merck, Germany, 2.5M); catalase (1 mg/ml, Sigma-Aldrich, Germany) was then added to the CFCS and incubated at 25°C for 1 h [12]. The antimicrobial activity of these CFCS was investigated using the agar well diffusion assay.
Identification of LAB species by 16S rRNA gene sequencing LAB strains with antimicrobial efficacy against all Shigella strains were identified by 16S rRNA gene sequencing. DNA extraction of strains was performed using the boiling method [20]. PCR was performed with primers 27F (5'-CTCGTTGCGGGACTTAA-3') and 1522R (5'-GCAGCAGTAGGGAATCTTC-3') (Bioneer, Korea) [21]. The reaction mixture consisted of 1.5 mM MgCl 2 , 0.2 mM dNTPs, 2.75 ml of genomic DNA, 5 ml 10X PCR buffer, 3 pmol of each primer and 3 U of Taq DNA polymerase (Jena Bioscience, Germany) in a final volume of 50 ml. The PCR protocol started with an initial denaturation at 95°C for 2 min, followed by 35 cycles of 94°C for 1 min, Table 1: Inhibitory activity of cell-free culture supernatants (CFCS) of Lactobacillus strains against Shigella strains 55°C for 1 min and 72°C for 10 min. PCR products were electrophoretically separated on agarose gel (1.5% w/v) and visualized by staining with safe stain (Yekta Tajhiz Azma, Iran). Finally, PCR products were sent for sequencing (Bioneer, Korea). Species of LAB were identified after comparison of the obtained sequences by nucleotidenucleotide BLAST (https://www.ncbi.nlm.nih.gov/blast).

Discussion
Shigellosis is usually a self-limiting infection, but in severe cases of the infection, antibiotic therapy may be required. Quinolones and cephalosporins are the drugs of choice. However, the worldwide emergence of antimicrobial resistance among Shigella species has limited the choice of antimicrobial agents for treating the infection. Resistance to different antibiotics such as sulfonamides, ampicillin, tetracyclines and co-trimoxazole has been described among Shigella species worldwide, and the causes of treatment failure can be attributed to these antibiotic resistances [13]. Because there are concerns about the increase in drug-resistant pathogenic bacteria, probiotic LAB are being used as a preventive treatment alternative [22]. Probiotic bacteria have been widely studied over the past decades in the prevention and treatment of diarrheal diseases.  [24] showed that Lb. rhamnosus GG has inhibitory activity against S. sonnei ATCC 25931. In addition, Zhihui Yu et al. [25] isolated Lb. plantarum S4-1 from naturally-fermented Chinese sauerkraut, and showed that this strain possesses inhibitory activity against S. flexneri CMCC. They suggested that Lb. plantarum S4-1 have potential as an excellent probiotic candidate for use in functional products. In the present study, when the CFCS of Lactobacillus was adjusted to pH 6.5 and treated with catalase, the antimicrobial activity disappeared. These results suggest that the production of bacteriocin-like compounds did not play a role in the mechanism of antimicrobial activity. The inhibition of Shigella strains appeared to be the result of organic acids or hydrogen peroxide production by the Lactobacillus strains. Several studies have previously shown that a pH-dependent mechanism was involved in the antimicrobial activity of Lactobacillus strains [26], [27].

Conclusions
Lactobacillus strains with good inhibitory activity against S. flexneri and S. sonnei were isolated from traditional Iranian yogurt and milk. These Lactobacillus strains may be useful as potential novel and effective probiotic strains for the prevention or treatment of diarrhea, but further in vitro and in vivo investigations on these strains are still required.

Notes Competing interests
The authors declare that they have no competing interests.